The Tox21 programs federal partners include the Environmental Protection Agency (EPA), the Food and Drug Administration (FDA) and NIH, with leadership from NCATS and the National Toxicology Program (NTP) at the National Institute of Environmental Health Sciences (NIEHS). These agencies work together to advance in vitro toxicological testing. The Tox21 Program is comprised of three NCATS teams: Systems Toxicology, Genomic Toxicology, and Computational Toxicology. The Systems Toxicology team has identified, developed, optimized, and/or screened more than 19 assays. Highlights range from performing 5 online screens, including caspase 3/7 assays in CHO and HepG2 cells, human pregnane X receptor in triplicate and acetylcholinesterase (AChE) enzyme-based assay with or without metabolic capability in single against the Tox21 10K compound collection and 5 online validation assays, including enzyme-based AChE assay with or without liver microsomes assays, against the LOPAC collection on the Tox21 robotic system. All of these assays were optimized and evaluated before moving to robotic online validation and online screening. AChE is the primary cholinesterase in the body that metabolizes a key neurotransmitter, acetylcholine. Inhibition of AChE activity can lead to neurotoxicity and known inhibitors include organophosphorus pesticides, chemical warfare agents, drugs, and various phytochemicals. In collaboration with the CFSAN/FDA, the Systems Toxicology team has screened two cell-based AChE assays with fluorescence and color metric readouts in a homogenous format against the Tox21 10K compound collection. In addition, enzyme-based AChE assays with or without liver microsomes have been developed and validated against a group of known AChE inhibitors that need metabolism to become active compounds. In addition, the team has worked with NTP, EPA and FDA on the mitochondria project and tested 34 compounds identified from the primary screening in the follow-up assays including reactive oxygen species (ROS), p53 and Nrf2/ARE (antioxidant response element), mitochondrial oxygen consumption, Parkin translocation, and larval development and ATP status in the C. elegans. Known mitochondrial complex inhibitors (e.g., rotenone) and un-couplers (e.g., chlorfenapyr), as well as potential novel complex inhibitors and un-couplers, were detected. The team has also optimized and validated in vitro co-culture angiogenesis assay in a 1536-well plate format by screening a drug collection contacting 2816 compounds in a high content imager. From the screen, many known angiogenesis inhibitors were identified such as topotecan, docetaxel, and bortezomib. Several potential novel angiogenesis inhibitors were also identified from this study including thimerosal and podofilox. Among the inhibitors, some compounds were proved to be involved in the hypoxia inducible factor-1-alpha (HIF-1) and the nuclear factor-kappa B (NF-B) pathways. The estrogen-related receptor alpha (ERR) is an orphan nuclear receptor (NR) that plays a role in energy homeostasis and controls mitochondrial oxidative respiration. In collaboration with NIEHS, we identified more than five agonist clusters including statin cluster based on compound structural similarity analysis (e.g., statins) after primary screening. We also performed follow-up assays (e.g., siRNA knockdown) we identified compounds that might act as endocrine disrupters through effects on ERR signaling. In addition, the team has identified a group of CAR activators and deactivators from the primary screening. Several follow-up studies including CAR translocation in human hepatocytes have been performed to confirm the compounds with CAR activity.